This project was designed to investigate the cellular and molecular pathways involved in the HIV regulatory effects of host and viral factors.Since its inception, the Immunopathogenesis Section has defined host factors involved in the pathogenesis of HIV disease, including the impact of the immune system on the propagation and/or control of virus replication. HIV replication in vivo represents, in part, a balance between the maintenance of an effective HIV-specific cellular immune response and the ability of HIV to replicate .The object of this project was to investigate the effect of the newly described immunoregulatory CD4+CD25+ subset of T cells on the host response to HIV infection. CD25+ CD4+ T cells isolated from the peripheral blood of HIV-infected donors expressed the cell surface and nuclear antigen phenotype characteristic of CD25+hi suppressor CD4+ T cells (CD45RA-/lo, elevated CTLA-4, CD103, CD122 and transcripton factor FoxP3 (+)); however, the frequency of this population was not elevated as compared to the frequency found in PBMC of HIV negative donors. Depletion of the CD25+ subset from memory CD4+ T cells enhanced, and re-addition suppressed, CD4+ T cell proliferation and IL-2 and IFN-g production in response to HIV p24 antigen, particularly in cells from individuals with CD4+ T cell counts >= 300/?L and low to undetectable levels of plasma viremia. As previously reported for CD25+hi suppressor cells, this effect was not significantly overcome by neutralization of IL-10 or TGF-b. The presence of CD25+CD4+ T cells in PBMC also inhibited production of IFN-g by CD8+ T cells in response to HIV peptides; this effect was seen with cells from most HIV-infected donors tested. Furthermore, CD25+CD4+T cell depletion resulted in elevated constitutive IFN-g production by CD8+ PBMC isolated from HIV-infected patients with moderate to high, but not those with low to undetectable, levels of plasma viremia, suggesting that CD25+CD4+ regulatory T cells may repress hyper-activated CD8+ T cells in vivo. Finally, the ability of effector (perforin (+)) CD8+ T cells to proliferate in response to autologous HIV super-infected target T cells was suppressed by the addition of CD25+, but not CD25-, CD4+ T cells. These data demonstrate that CD25+CD4+ T cells can exert dramatic immunosuppressive effects on both CD4+ and CD8+ HIV-specific T cell immune responses in vitro and imply that CD25+CD4+ T cells may interfere with HIV-specific immune responses in vivo. CD25+CD4+ T cells were also found to suppress soluble and cell contact-mediated suppression of HIV replication by CD8+ T cells from certain HIV+ donors. Preliminary studies for assessing the effect of CD25+ cell depletion on immune responses and viral replication in SIV-infected macaques are currently underway. RNA microarray analyses and iRNA technology are currently being utilized to determine the mechanism(s) by which CD25+CD4+ regulatory T cells suppress HIV-specific immune responses and non-lytic anti-viral activities of CD8+ T cells. In this regard, we have successfully designed a lentiviral vector capable of efficiently transducing primary T cells and expressing siRNAs directed against gene products of interest. HIV envelope protein (gp160) was found to stimulate the production of infectious virus from HIV-infected donor's resting CD4+ T cells, a major cellular reservoir of HIV, without inducing markers of cellular activation, division or apoptosis. Using an in vitro human lymphoid tissue (tonsil) culture model, we have found that the lymphoid tissue microenvironment supports productive HIV (X4>>R5 strains) infection of phenotypically resting CD4+ T cells( lack both surface and nuclear markers of classical T cell activation. X4 HIV-exposed purified resting CD4+ T cells produced HIV mRNA, p24 protein and infectious virus while maintaining a resting, non-dividing T cell phenotype when re-introduced into the microenvironment of autologous lymphoid tissue, but not when cultured alone. Pro-inflammatory cytokine antagonists and the immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor (TGF)-b, but not a potent inhibitor of T cell division/ full activation (mycophenoloic acid), effectively suppress X4 HIV production from resting CD4+ T cells in the lymphoid tissue demonstrating a role for endogenous proinflammatory cytokines in driving HIV production from this cellular population. These data repudiate the paradigm that HIV production in T cells requires classical T cell activation and demonstrate that the lymphoid tissue is an ideal microenvironment for suboptimal cellular activation leading to HIV replication in phenotypically resting CD4+ T cells. .